Venom samples were analysed using 15% (m/v) polyacrylamide gels in the presence of SDS (SDS-PAGE) un-der non-reducing and reducing conditions according to Laemmli. 8 (link) Proteins in gels were visualized by Page-Blue TM (Thermo Scientific, USA) as instructed by the manufacturer. Molecular mass standards were from Fermentas (Lithuania).
Molecular mass standards
Manufactured by Thermo Fisher Scientific
Sourced in United States
Molecular mass standards are laboratory products used to determine the molecular weight of biomolecules such as proteins, peptides, and nucleic acids. They provide a reference for calibrating and verifying the performance of various analytical techniques, including gel electrophoresis and mass spectrometry.
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4 protocols using molecular mass standards
1
SDS-PAGE Analysis of Venom Proteins
2
Gelatin-SDS-PAGE Analysis of Extracellular Proteases
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The extracellular proteolytic activity (50 µg protein/ slot) was determined using 10% SDS-PAGE with 0.1% copolymerized gelatin as substrate (Heussen and Dowdle 1980). Electrophoresis was performed at 120V, 4°C for 90 min, and the gels were then incubated in 2.5% Triton X-100 for 1 h, washed in distilled water and incubated for 24 h in one of the following pH buffers: 10 mM sodium citrate (pH 3.0), 50 mM sodium phosphate (pH 5.0 and pH 7.0) and 20 mM glycine-NaOH (pH 9.0 and pH 11.0). The effect of the temperature upon the enzyme activity was assessed by incubating the gels at 4°C, 27°C, 37°C and 56°C. The detection of proteolytic bands was performed by staining the gels for 2 h with 0.2% Coomassie Brilliant Blue R-250 in methanolacetic acid-water (50:10:40, v/v/v) and destaining in the same solvent. The molecular mass of the proteases was calculated by comparison with the mobility of molecular mass standards (Fermentas). The gels were dried, scanned and the densitometric analysis was digitally processed using Image J public domain software.
3
Polypeptide Pattern Analysis of WKB Extract
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The polypeptide pattern of the WKB extract was analyzed by SDS-PAGE [24 (link)] and compared to a commercial product Phase 2® WKB Extract (NOW Foods, Bloomingdale, USA). In brief, samples were dissolved in Tris-HCl buffer (1 mol/L, pH 6.8) and loaded onto the gel with 0.2 mg soluble protein. Molecular mass standards (10–180 kDa; Thermo Fisher, USA) were also loaded in a separate well on each gel. Electrophoresis was performed in 62.5 mmol/L Tris-HCl buffer with 3.4 mmol/L SDS for 2 h (Bio-Rad, USA). The electrophoresis results were visualized by Coomassie brilliant blue staining and analyzed with Tanon 5200 Digital Gel Image System (Tanon, China).
4
Salivary Glycoprotein Profiling by SDS-PAGE and Lectin Blotting
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The pooled salivary proteins from HV, BB, BC-I, and BC-II subjects of the retrospective cohort were analyzed by SDS-PAGE and subsequently lectin blotting as previously described (Qin et al., 2013 (link), Zhong et al., 2015 (link)). Briefly, For SDS-PAGE, samples were boiled for 4 min at 100 °C mixed with 5 × loading buffer, and run on a 10% polyacrylamide resolving gel and a 3% stacking gel. Molecular mass standards (Thermo Scientific, Waltham, USA) were run with all gels. Some gels were then stained directly with alkaline silver. For lectin blotting, the proteins in gels were then transferred to a PVDF membrane (Immobilon-P; Millipore Corp., Bedford, MA, U.S.A.) with a wet transfer unit (Hoefer Scientific) for 1.5 h at 32 mA. After transfer, the membranes were washed twice with TTBS (150 mM NaCl, 10 mM Tris-HCl, 0.05% v/v Tween20, pH 7.5) and then blocked for 1 h with Carbo-Free Blocking Solution (Vector, Burlingame, CA) at room temperature. The membranes were then washed again and incubated with Cy5 (GE Healthcare, Buckinghamshire, UK) labeled lectins (2 μg/mL in Carbo-Free Blocking Solution) with gentle shaking overnight at 4 °C in the dark. The membranes were then washed twice each for 10 min with TTBS and scanned by red fluorescence channel (635 nm excitation/650LP emission) with the voltage of 800 PMT using a phosphor imager (Storm 840, Molecular Dynamics).
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